Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport
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Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17β-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 μM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 μM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 μM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 μM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17β-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.Keywords:
Efflux
Glucuronide
Mediated transport
Abcg2
Much evidence has demonstrated that a number of ATP-binding cassette (ABC) efflux transporters including P-glycoprotein (PGP), the multidrug resistance-associated proteins (MRPs) and the breast cancer resistance protein (BCRP) are highly expressed in placental tissues and are believed to profoundly limit the passage of therapeutic or toxic xenobiotics to the fetus. Recent studies indicate that the oral hypoglycemic glyburide does not cross the human placenta to an appreciable extent. Our objective was to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus. Thus, [3H]-glyburide transport was examined in BCRP, PGP, MRP1, MRP2 and MRP3 over-expressing cell lines in the presence or absence of specific inhibitors. Our results demonstrated significant increases in the intracellular accumulation of [3H]-glyburide in BCRP and MRP3 over-expressing cells in the presence of the inhibitors novobiocin and indomethacin, respectively. PGP inhibition with verapamil or MRP inhibition with indomethacin did not affect [3H]-glyburide accumulation in the PGP or MRP2 over-expressing cell lines and only limited changes were seen in the MRP1 over-expressing cell line. On the other hand, glyburide was found to significantly inhibit MRP1-, MRP2- and MRP3-mediated efflux of 5-carboxyfluorescein diacetate and PGP-mediated transport of rhodamine 123. Our evidence is the first to clearly indicate that glyburide is preferentially transported by BCRP and MRP3.
Abcg2
Efflux
Transplacental
Rhodamine 123
P-glycoprotein
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The purpose of this study is to characterize how overexpression of an efflux transporter and an UDP-glucuronosyltransferase (UGT) affects the cellular kinetics of glucuronidation processes.A new MDCK II cell line overexpressing both MRP2 and UGT1A1 (MDCKII-UGT1A1/MRP2 cells) was developed and used to determine how overexpression of an efflux transporter affects the kinetics of cellular flavonoid glucuronide production. The results showed that most model flavonoids (from a total of 13) were mainly metabolized into glucuronides in the MDCKII-UGT1A1/MRP2 cells and the glucuronides were rapidly excreted. Flavonoids with three or fewer hydroxyl group at 7, 3' or 6 hydroxyl group were also metabolized into sulfates. Mechanistic studies using 7-hydroxylflavone showed that its glucuronide was mainly (90%) effluxed by BCRP with a small (10%) but significant contribution from MRP2. Maximal velocity of glucuronide production MDCK-MRP2/UGT1A1 cells showed a fairly good correlation (R(2) >0.8) with those derived using UGT1A1 microsomes, but other kinetic parameters (e.g., Km ) did not correlate.Overexpression of a second efficient efflux transporter did not significantly change the fact that BCRP is the dominant transporter for flavonoid glucuronide nor did it diminish the influence of the efflux transporter as the "gate keeper" of glucuronidation process.
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Glucuronide
Glucuronosyltransferase
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The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2) and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP) function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6)-carboxy-2′,7′-dichloroflourescein (CDF) was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.
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Caco-2
Isoflavonoid
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Flurbiprofen is a nonsteroidal anti-inflammatory drug used as a racemic mixture. Although glucuronidation is one of its elimination pathways, the role of UDP-glucuronosyltransferase (UGT) in this process remains to be investigated. Thus, the kinetics of the stereoselective glucuronidation of racemic (R,S)-flurbiprofen by recombinant UGT isozymes and human liver microsomes (HLMs) were investigated, and the major human UGT isozymes involved were identified. UGT1A1, 1A3, 1A9, 2B4, and 2B7 showed glucuronidation activity for both (R)- and (S)-glucuronide, with UGT2B7 possessing the highest activity. UGT2B7 formed the (R)-glucuronide at a rate 2.8-fold higher than that for (S)-glucuronide, whereas the other UGTs had similar formation rates. The glucuronidation of racemic flurbiprofen by HLMs also resulted in the formation of (R)-glucuronide as the dominant form, which occurred to a degree similar to that by recombinant UGT2B7 (2.1 versus 2.8). The formation of (R)-glucuronide correlated significantly with morphine 3-OH glucuronidation (r = 0.96, p < 0.0001), morphine 6-OH glucuronidation (r = 0.91, p < 0.0001), and 3′-azido-3′-deoxythymidine glucuronidation (r = 0.85, p < 0.0001), a reaction catalyzed mainly by UGT2B7, in individual HLMs. In addition, the formation of both glucuronides correlated significantly (r = 0.99, p < 0.0001). Mefenamic acid inhibited the formation of both (R)- and (S)-glucuronide in HLMs with similar IC50 values (2.0 and 1.7 μM, respectively), which are close to those in recombinant UGT2B7. In conclusion, these findings suggest that the formation of (R)- and (S)-glucuronide from racemic flurbiprofen is catalyzed by the same UGT isozyme, namely UGT2B7.
UGT2B7
Glucuronide
Glucuronosyltransferase
Flurbiprofen
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Enniatins are cyclic hexadepsipeptides produced by various fungi, known to have ionophoric, antibiotic and insecticidal activity. The aim of the present study was to evaluate the intestinal absorption characteristics of enniatin B 1 (ENN-B 1 ). Using the human intestinal Caco-2 cell line, we found that the permeability of ENN-B 1 in the basolateral to apical direction was 6.7× higher as compared to the permeability in the opposite direction, indicating involvement of apically located transporters. Transport of ENN-B 1 in the apical to basolateral direction was increased significantly upon treatment of Caco-2 cells with the P-glycoprotein (Pgp) inhibitor verapamil and the multidrug resistance-associated protein 2 (MRP2) inhibitor MK571, but only weakly and not significantly after treatment with the breast cancer resistance protein (BCRP) inhibitor fumitremorgin C. Additionally, MDCK II cells over-expressing Pgp, MRP2 or BCRP, showed reduced sensitivity towards ENN-B 1 . These data demonstrate for the first time that ENN-B 1 is a substrate of MRP2 and suggests that Pgp, MRP2 and possibly BCRP are involved in transport of ENN-B 1 across the intestine.
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P-glycoprotein
Caco-2
Efflux
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ATP-binding cassette (ABC) multidrug transporters are drug efflux pumps located in the plasma membrane that utilize the energy of ATP hydrolysis to extrude a wide spectrum of endogenous and exogenous compounds from cells, including numerous (anticancer) drugs and/or their metabolites. The studies described in this thesis focus on the pharmacological functions of the ABC transporters: P-glycoprotein (P-gp/ABCB1), the Multidrug Resistance Proteins 2 and 3 (MRP2/ABCC2 and MRP3/ABCC3) and the Breast Cancer Resistance Protein (BCRP/ABCG2). Most results presented in this thesis were obtained by studying single and combination ABC multidrug transporter knockout mice. As ABC multidrug transporters do not only have very broad, but also substantially overlapping substrate specificities, they can often partially, or sometimes even fully, compensate for the loss of each other. Combination ABC drug transporter knockout mice are therefore invaluable tools to study the separate roles and functional overlap of ABC multidrug transporters. We generated and characterized combination P-gp/Mrp2 knockout mice and used these to assess the distinct roles of P-gp and Mrp2 in the pharmacokinetics of the anticancer drug paclitaxel. Although paclitaxel is an excellent P-gp substrate, Mrp2 was found to almost exclusively mediate the excretion of paclitaxel from the liver into the bile, whereas P-gp had little effect. This finding is especially interesting because Mrp2 was thus far thought to mainly affect organic anionic drugs in vivo. However, we show that Mrp2 can also be a major determinant of the pharmacokinetic behavior of highly lipophilic anti-cancer drugs, even in the presence of other efficient transporters. P-gp and BCRP combination knockout mice enabled us to demonstrate that both multidrug transporters act in concert at the blood-brain barrier in restricting the brain penetration of the novel tyrosine kinase inhibitor anticancer drugs dasatinib and sorafenib. Brain penetration of dasatinib was primarily restricted by P-gp, whereas loss of BCRP had no effect. However, when both transporters were absent a disproportionate increase in brain penetration of dasatinib was observed. In contrast, for sorafenib it was the other way around, i.e. absence of P-gp had no effect while BCRP deficiency resulted in markedly elevated brain levels. Again, simultaneous loss of both transporters resulted in a highly increased brain penetration. When we combined dasatinib with the dual P-gp and BCRP inhibitor elacridar we found that the brain penetration in wild-type mice could be increased to P-gp/BCRP knockout levels. These findings might be clinically relevant for patients with intracranial tumors, as concomitant administration of an inhibitor of P-gp and ABCG2 with dasatinib, sorafenib and possibly other tyrosine kinase inhibitors might result in better therapeutic responses in these patients. In conclusion, the studies described in this thesis demonstrate the power of combination ABC multidrug transporter knockout mouse models to study the pharmacological functions of ABC multidrug transporters. We expect that combination ABC transporter knockout mice will be extensively used as preclinical research tools.
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P-glycoprotein
Efflux
Knockout mouse
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Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17β-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.
Abcg2
Efflux
Organic anion-transporting polypeptide
Organic anion
Estrone sulfate
Multidrug Resistance-Associated Proteins
ABCC1
Probenecid
Renal physiology
Transport protein
Paracellular transport
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Overexpression of ABC drug transporters can cause multidrug resistance (MDR) in cancer cells, which is a major obstacle in the success of cancer chemotherapy. Our study revealed a correlation between the expression of invasive breast cancer resistance-associated proteins, such as P-glycoprotein (ABCB1), MRP2 (ABCC2), BCRP (ABCG2) in tumor cells and pathologic response to neoadjuvant chemotherapy. The response to neoadjuvant chemotherapy was shown to be associated with a lack of BCRP expression in tumor cells. The pathologic tumor response was correlated with the presence of positive MRP2 expression and the expression level of P-glycoprotein in cells of invasive breast cancer.
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P-glycoprotein
Multidrug Resistance-Associated Proteins
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P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) are clinically relevant efflux transporters implicated in the oral absorption of many food and drug substrates. Here, we hypothesised that food intake could influence protein and mRNA intestinal expression of P-gp/abcb1a, BCRP/abcg2, and MRP2/abcc2 differently in male and female Wistar and Sprague Dawley rats. To test this hypothesis, we used enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) to quantify the protein and mRNA intestinal expression of these transporters, respectively. Our study found food and sex differences in P-gp expression, whereby in the fed state P-gp expression decreased in male Wistar rats, but P-gp expression increased in females. In the fed state, BCRP expression increased in both male and female Wistar rats, compared with the fasted state. In contrast, no sex differences or food effect differences were seen in Sprague Dawley rats for P-gp and BCRP expression. On the other hand, in the fed state, MRP2 expression was higher in male and female Wistar and Sprague Dawley rats when compared with the fasted state. Sex differences were also observed in the fasted state. Overall, significant strain differences were reported for P-gp, BCRP and MRP2 expression. Strong to moderate positive linear correlations were found between ELISA and PCR quantification methods. ELISA may be more useful than PCR as it reports protein expression as opposed to transcript expression. Researchers must consider the influence of sex, strain and feeding status in preclinical studies of P-gp, BCRP and MRP2 drug substrates.
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Efflux
P-glycoprotein
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Intestinal ATP binding cassette (ABC) transporters may affect the bioavailability and effectiveness of orally administered drugs. Available studies on regional expression of intestinal efflux transporters were done with selected intestinal segments only and inconsistent with regard to the variability of transporter expression and the course of expression along the intestine. For an evaluation of the consistency between mRNA and protein expression, relative expression levels of P-glycoprotein (Pgp; ABCB1), breast cancer resistance protein (Bcrp; ABCG2), and multidrug resistance-associated protein (Mrp) 2 (ABCC2) were determined using quantitative real-time-polymerase chain reaction and Western blot in rat intestinal segments from duodenum, jejunum, ileum, and colon. In addition, the protein expression of Pgp, Bcrp, and Mrp2 from the entire rat intestine was studied by a complete 3-cm segmentation to evaluate the predictive power of expression analyses from selected intestinal segments. Pgp showed an increase from proximal to distal regions, Bcrp showed an arcuate pattern with highest expression toward the end of small intestine, and Mrp2 decreased along the intestinal axis from proximal to distal parts. No gender specific differences could be observed. Regarding the concordance of mRNA and protein expression, Pgp and Bcrp mRNA samples allow good estimations about the corresponding protein expression (for Pgp limited to the mdr1a isoform), but for Mrp2, pronounced deviation could be observed. All transporters showed considerable intra- and interindividual variability, especially at the protein level, making it problematic to take transporter expressions of small sections exemplary for general assumptions on intestinal abundances.
Abcg2
P-glycoprotein
Efflux
Multidrug Resistance-Associated Proteins
Jejunum
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